Anti-cd95l antibody

ABSTRACT

The present invention relates to a specific CD95L antibody and to the use thereof in the treatment or diagnosis of diseases involving CD95L-induced signalling, e.g. cancer diseases.

This application is a continuation of PCT/EP2016/072757, filed Sep. 23,2016; which claims the priority of EP 15186468.3, filed Sep. 23, 2015.The contents of the above applications are incorporated herein byreference in their entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with thespecification as an ASCII formatted text file via EFS-Web with a filename of Sequence Listing.txt with a creation date of Mar. 19, 2018, anda size of 96.5 kilobytes. The Sequence Listing filed via EFS-Web is partof the specification and is hereby incorporated in its entirety byreference herein.

DESCRIPTION

The present invention relates to a specific CD95L antibody and to theuse thereof in the treatment or diagnosis of diseases involving CD95Linduced signalling, e.g. cancer disease.

The field of the present invention can be seen in particular in theimprovement of cancer therapy and cancer diagnostic.

The present invention provides a monoclonal antibody that specificallybinds to a linear epitope of CD95L and is capable to inhibit CD95Linduced signalling. It was surprisingly found, that the antibody of theinvention inhibits signalling induced by CD95/CD95L with higher efficacythan previously known antagonists of CD95/CD95L, in particular otheranti-CD95L antibodies, soluble CD95 molecules or fusion proteins likeAPG101.

Thus, a first aspect of the invention is a monoclonal anti-CD95Lantibody specifically binding to an epitope of human CD95L comprisingthe amino acid sequence NSKYP.

The binding epitope comprises the amino acid sequence RNSKYP, preferablyRNSKYPQ, which is found in CD95L of many different species. Preferably,the antibody binds to a linear epitope of CD95L comprising the aminoacid sequence RNSKYPQD and/or RNSKYPED. Human CD95L as well as CD95Lfrom monkeys, e.g. Macaca fascicularis, comprises the epitope RNSKYPQD,while mouse CD95L from mus musculus comprises the epitope RNSKYPED.According to a preferred aspect of the invention, the antibody iscapable to specifically bind to CD95L derived from different species,e.g. human, monkey and mouse. It is preferred that the antibodyspecifically binds to human, and at least one of monkey (e.g. macacafascicularis) and mouse (mus musculus) CD95L, more preferably to human,monkey and mouse CD95L.

The term “antibody” particularly refers to molecules comprising at leastone immunoglobulin heavy chain and at least one immunoglobulin lightchain. Each heavy and light chain may comprise a variable and a constantdomain. The antigen binding site may be formed from the variable domainsof a heavy chain and a light chain. A variable region (also referred toas variable domain) comprises complementarity determining regions(CDRs), e.g., a CDR1, a CDR2 and a CDR3 region and framework regions(FRs) flanking the CDRs.

The term “complementarity determining region” is readily understood bythe skilled person (see, e.g., Harlow and Lane (EDS.), Antibodies: ALaboratory Manual, CSHL Press, Cold Spring Harbour, N.Y., 1988) andrefers to the stretches of amino acids within the variable domain of anantibody that primarily make contact with the antigen and determineantibody specificity. This region is also known as the hypervariableregion.

The present invention encompasses both full length immunoglobulin andfunctional immunoglobulin fragments like Fab, Fab′, F(ab′)2 fragments,Fv fragments, diabodies, single-chain antibody molecules andsingle-domain antibodies. Also other fragments are included as long asthey exhibit the desired capability of binding to an epitope comprisingamino acids 214-219 of human CD95L, comprising the amino acid sequence“RNSKYP”, preferably amino acids 214-220, comprising the amino acidsequence “RNSKYPQ” and most preferably amino acids 214-221, comprisingthe amino acid sequence “RNSKYPQD”. For a review of certain antibodyfragments, see Hudson et al., Nat. Met. 9: 129-134 (2003).

“Diabodies” are antibody fragments with two antigen-binding sites thatmay be bivalent or bispecific (see, e.g., Hudson et al., 2003).“Single-chain antibodies” are antibody fragments comprising all or aportion of the heavy chain variable domain, or all or a portion of thelight chain variable domain of an antibody. Antibody fragments can bemade by various techniques, including but not limited to proteolyticdigestion of an intact antibody as well as production by recombinanthosts (e.g., E-coli or phage) as described herein.

Also encompassed by the present invention are human antibodies. The term“human antibody” is meant to encompass any fully human or humanizedantibodies. Human antibodies may be prepared from genetically engineeredanimals, e.g., animals comprising a xenogeneic immune system or fromantibody display libraries according to known techniques. Humanantibodies are described generally in Van Dijk and Van De Winkel (Car.Opin. Pharmacol. 5: 368-74 (2001)) and Lonberg (Car. Opin. Immunol. 20:450-459 (2008)).

Humanized antibodies may be prepared by humanization of monoclonalantibodies derived from other species (e.g. mouse, rat, rabbit)according to known techniques. Typically, a non-human antibody ishumanized to reduce immunogenicity to humans, while retaining thespecificity and affinity of the parental non-human antibody. Humanizedantibodies and methods of making them are reviewed, e.g. in Alamagro andFransson, Front. Biosci. 13: 1619-1633 (2008).

The antibodies of the present invention are characterized in that theyspecifically bind to an epitope of CD95L comprising the amino acidsequence RNSKYP, preferably RNSKYPQ and more preferably RNSKYPQD ofhuman CD95L. This binding epitope has been shown to be unique for itssuitability to inhibit CD95L-induced signalling. Antibodies binding tothis epitope directly compete with the binding of CD95L to the CD95receptor. Next to apoptosis, the corresponding receptor CD95 mediates,depending on the tissue and condition non-apoptotic signals (such asNF-kB, MAPK or PI3K), that promote inflammation, contribute tocarcinogenesis and modulate immunoncological parameters (e.g. tumourinfiltrating T-cell populations). All of these activities arepotentially inhibited by antibodies described herein.

The term “bind” or “binding” of an antibody means an at least temporaryinteraction or association with or to a target antigen, i.e. human CD95Lcomprising fragments thereof containing an epitope described herein. Incertain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g. 10⁻⁹ Mto 10¹³M). Methods for determining the Kd value are known to the personskilled in the art.

In one embodiment, Kd is measured by a radio-labelled antigen bindingassay (Radioimmunoassay, RIA) performed with the Fab version of anantibody of interest and its antigen.

According to another embodiment, Kd is measured using surface plasmonresonance assays with immobilized antigen. According to anotherembodiment Kd is measured by a quartz crystal microbalance (QCM) withimmobilized antigen. According to a preferred embodiment of the presentinvention, the antibodies are human monoclonal antibodies directedagainst an epitope of human CD95L as described herein.

The antibodies of the invention may be of various immunoglobulin (Ig)types, for example of the IgA-, IgD-, IgE-, IgG- or IgM-type, preferablyof the IgG- or IgM-type including but not limited to the IgG1-, IgG2-,IgG3-, IgG4-, IgM1 and IgM2-type. In one preferred embodiment theantibody is of the IgG1type.

Preferably, the antibodies of the invention are monoclonal antibodies.

In certain embodiments of the present invention, the antibody maycomprise specific heavy chain complementarity determining regions CDRH1,CDRH2 and/or CDRH3. The CDR sequences described herein are numberedusing the Kabat scheme.

In one embodiment, the antibody comprises a heavy chain comprising:

a heavy chain complementarity determining region 1 (CDRH1) having theamino acid sequence as shown in SEQ ID NO: 1 or 11,

a heavy chain complementarity determining region 2 (CDRH2) having theamino acid sequence as shown in SEQ ID NO: 2 or 12, and/or

a heavy chain complementarity determining region 3 (CDRH3) having theamino acid sequence as shown in SEQ ID NO: 3 or 13.

The antibody according to the invention may also comprise specific lightchain complementarity determining regions CDRL1, CDRL2 and/or CDRL3.Accordingly, in one embodiment, the antibody comprises a light chaincomprising:

a light chain complementarity determining region 1 (CDRL1) having theamino acid sequence as shown in SEQ ID NO: 4 or 14,

a light chain complementarity determining region 2 (CDRL2) having theamino acid sequence as shown in SEQ ID NO: 5 or 15, and/or

a light chain complementary determining region 3 (CDRL3) having theamino acid sequence as shown in SEQ ID NO: 6 or 16.

In a preferred embodiment, the antibody comprises a specific combinationof CDRs within one heavy chain and/or within one light chain.Accordingly, a particularly preferred antibody of the present inventioncomprises

a heavy chain including a CDRH1 as shown in SEQ ID NO: 1 or 11,

a CDRH2 as shown in SEQ ID NO: 2 or 12, and

a CDRH3 as shown in SEQ ID NO: 3 or 13, and

a light chain including

a CDRL1 as shown in SEQ ID NO: 4 or 14,

a CDRL2 as shown in SEQ ID NO: 5 or 15, and

a CDRL3 as shown in SEQ ID NO: 6 or 16.

Preferred is an antibody comprising a heavy chain comprising CDRH1 asshown in SEQ ID NO: 1, CDRH2 as shown in SEQ ID NO: 2 and CDRH3 as shownin SEQ ID NO: 3 and a light chain comprising CDRL1 as shown in SEQ IDNO: 4, CDRL2 as shown in SEQ ID NO: 5 and CDRL3 as shown in SEQ ID NO:6.

Also preferred is an antibody comprising a heavy chain comprising CDRH1as shown in SEQ ID NO: 11, CDRH2 as shown in SEQ ID NO: 12 and CDRH3 asshown in SEQ ID NO: 13 and a light chain comprising CDRL1 as shown inSEQ ID NO: 14, CDRL2 as shown in SEQ ID NO: 15 and CDRL3 as shown in SEQID NO: 16.

In a preferred embodiment of the invention, the anti-CD95L antibodycomprises a heavy chain variable region (VH) as shown in SEQ ID NO: 7 or17 or a sequence having a sequence identity of at least 90% over thewhole heavy chain variable region, preferably at least 95% sequenceidentity, more preferably at least 96%, 97%, 98% or 99% sequenceidentity. Furthermore, the antibody of the invention preferablycomprises a light chain variable region (VL) as shown in SEQ ID NO: 8 or18 or a sequence having a sequence identity of at least 90% over thewhole light chain variable region, preferably at least 95% sequenceidentity, more preferably at least 96%, 97%, 98% or 99% sequenceidentity. Particularly preferred are antibodies comprising a heavy chainvariable region as shown in SEQ ID NO: 7 or 17 and a light chainvariable region as shown in SEQ ID NO: 8 or 18.

Preferably, an antibody of the invention comprises a heavy chainvariable region as shown in SEQ ID NO: 7 and a light chain variableregion as shown in SEQ ID NO: 8 or a heavy chain variable region asshown in SEQ ID NO: 17 and a light chain variable region as shown in SEQID NO: 18.

According to a particularly preferred embodiment of the invention, theantibody of the invention comprises a heavy chain comprising the aminoacid sequence as shown in SEQ ID NO: 9 or 19, or an amino acid sequencehaving a sequence identity of at least 90% thereto over the whole heavychain amino acid sequence, and a light chain comprising an amino acidsequence as shown in SEQ ID NO: 10 or 20, or an amino acid sequencehaving a sequence identity of at least 90% thereto over the whole lengthof the light chain amino acid sequence. The sequence identity of theheavy chain and the light chain amino acid sequence is preferably atleast 95%, more preferably at least 96%, 97%, 98% or 99% to thesequences shown in SEQ ID NO: 9, 10, 19 and 20. Most preferred is anantibody comprising the heavy chain amino acid sequence as shown in SEQID NO: 9 and the light chain amino acid sequence as shown in SEQ ID NO:10 as well as an antibody comprising the heavy chain amino acid sequenceas shown in SEQ ID NO: 19 and the light chain amino acid sequence asshown in SEQ ID NO: 20.

Preferred Humanized Antibodies of the Invention

To determine the epitope on human CD95L recognized by the antibody,chemically prepared arrays of short peptides derived from the amino acidsequence of human CD95L can be used to locate and identify antibodyepitopes (Reinike W., Methods Mol. Biol., 2004, 248: 443-63). A furthermethod to map the epitopes in human CD95L bound by the antibodies of theinvention comprises Snaps/SELDI (Wang et al., Int. J. Cancer, 2001, June15, 92(6): 871-6) or a routine cross-blocking assay such as described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988) can be performed.

As mentioned above, the antibodies of the invention show advantageousproperties with respect to their binding specificity and biologicalactivity, in particular with respect to their capability to inhibitCD95L-induced signalling.

The antibodies of the present invention may be coupled to a heterologousgroup, e.g., a label or an effector group.

An antibody conjugate comprising an antibody of the invention coupled toan effector group is particularly suitable for therapeutic applications.As used herein, the term “effector group” refers to a therapeutic group,a toxin, a cytotoxic group, an antigen or other effector group known inthe art.

An antibody conjugate comprising an antibody of the invention coupled toa label group is particularly suitable for diagnostic applications. Asused herein, the term “label group” refers to a detectable marker, e.g.,a radiolabelled amino acid or biotin moiety, a fluorescent marker, anenzyme or any other type of marker which is known in the art.

The invention also relates to a nucleic acid molecule encoding theantibody as disclosed above. The term “nucleic acid molecule”encompasses DNA, e.g., single- or double-stranded DNA or RNA. The DNAmay be of genomic, cDNA or synthetic origin, or a combination thereof.The nucleic acid molecule of the invention may be in operative linkageto an expression control sequence, i.e. to a sequence which is necessaryto effect the expression of coding nucleic acid sequences. Suchexpression control sequences may include promoters, enhancers, ribosomalbinding sites and/or transcription termination sequences. Specificexamples of suitable expression control sequences are known in the art.

According to a preferred embodiment, the invention is directed to anisolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of

-   -   (a) a nucleic acid sequence encoding an antibody, or a        functional fragment thereof as defined above,    -   (b) a nucleic acid sequence complementary to any one of the        sequences in (a), and    -   (c) a nucleic acid sequence capable of hybridizing to (a) or (b)        under stringent conditions.

According to a particularly preferred embodiment of the invention, anucleic acid molecule comprises a sequence encoding the amino acidsequence of the variable region of the heavy chain and a sequenceencoding the amino acid sequence of the variable region of the lightchain of the antibody. In an alternative embodiment, a combination oftwo nucleic acid molecules is provided, wherein one nucleic acidmolecule encodes the amino acid sequence of the light chain of theantibody and the other nucleic acid molecule encodes the amino acidsequence of the heavy chain of the antibody. A preferred nucleic acidmolecule of the invention is an isolated nucleic acid moleculecomprising a nucleic acid sequence as shown in any one of SEQ ID NOs:21-24. For example, an isolated nucleic acid molecule may comprise thenucleic acid sequences as shown in SEQ ID NOs: 21 and 22 or the nucleicacid sequences as shown in SEQ ID NOs: 23 and 24.

The term “hybridizing under stringent conditions” means that two nucleicacid fragments hybridize with one another under standardizedhybridization conditions as described, for example in Sambrook et al.,“Expression of cloned Genes in E. coli” in Molecular Cloning: ALaboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York,USA. Such conditions are, for example, hybridization in 6.0×SSC (SalineSodium Citrate) at about 45° C. followed by a washing step with 2.0×SSCat 50° C., preferably 2.0×SSC at 65° C. or 0.2×SSC at 50° C., preferably0.2×SSC at 65° C.

The nucleic acid molecule of the invention may be located on a vectorwhich may additionally contain a replication origin and/or a selectionmarker gene. Examples of vectors are e.g. plasmids, cosmids, phages,viruses etc. Thus, a further embodiment of the invention is a vectorcomprising a nucleic acid sequence as disclosed herein. Preferably, thevector is an expression vector. Said vector may, for example, be aphage, plasmid, viral or retro viral vector. Retro viral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementinghosts/cells.

The nucleic acid molecules of the invention may be joined to a vectorcontaining selectable markers for propagation in a host. Generally, aplasmid vector is introduced in a precipitate such as a calciumphosphate precipitate or rubidium chloride precipitate or in a complexwith a charged lipid or in carbon-based clusters such as fullerenes.Should the vector be a virus, it may be packed in vitro using anappropriate packaging cell line prior to application to host cells.

Preferably, the vector of the invention is an expression vector, whereinthe nucleic acid molecule is operatively linked to one or more controlsequences allowing the transcription and optionally expression inprokaryotic and/or eukaryotic host cells. Expression of said nucleicacid molecule comprises transcription of the nucleic acid molecule,preferably into a translatable mRNA. Regulatory elements ensuringexpression in eukaryotic cells, preferably mammalian cells, arewell-known to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers. Expression vectors derived fromviruses such as retrovirus, vaccina virus, adeno-associated virus,herpes virus or bovine papilloma virus may be used for delivery of thepolynucleotides or vector of the invention into targeted cellpopulation. Methods which are well-known to those skilled in the art canbe used to construct recombinant viral vectors; see for example thetechniques described in Sambrook, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press (2001, 3^(rd) edition), N.Y.and Ausubel, Current Protocols in Molecular Biology, Green PublishingAssociates and Wiley Interscience, N.Y. (1994). Alternatively, thenucleic acid molecules of the invention can be reconstituted intoliposomes for delivery to target cells.

Further, the invention refers to a host which comprises the nucleic acidmolecule or the vector as described above. The nucleic acid molecule orthe vector may be introduced into the host by transformation,transfection or transduction according to any method known in the art.

Said host may be a prokaryotic or eukaryotic cell or a non-humantransgenic animal. The nucleic acid or vector of the invention which ispresent in the host may either be integrated into the genome of the hostor it may be maintained extrachromosomally. In this respect, it is alsoto be understood that the nucleic acid molecule of the invention can beused for “gene targeting” and/or “gene replacement”, for restoring amutant gene or for creating a mutant gene via homologous recombination;see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990),4712-4716; Joyner, Gene Targeting, A Practical Approach, OxfordUniversity Press.

The host can be any prokaryotic or eukaryotic cell such as a bacterial,insect, fungal, plant, animal, mammalian or preferably a human cell. Thetransformed hosts can be grown in fermenters and cultured according totechniques known in the art to achieve optimal cell growth. Theantibodies, antibody fragments or derivatives thereof of the inventioncan then be isolated from the growth medium, cellular lysates orcellular membrane fractions. The isolation and purification of themicrobially or otherwise expressed antibodies, antibody fragments orderivatives thereof of the invention may be by any conventional means,such as for example preparative chromatographic separations andimmunological separations such as those involving the use of monoclonalor polyclonal antibodies.

According to one embodiment of the invention, the host is a human,bacteria, animal, fungal, amphibian or plant cell. Preferred animalcells include but are not limited to Chinese hamster ovary (CHO) cells,baby hamster kidney (BHK) cells, monkey kidney cells (COS), mouseembryonic fibroblast cells (NIH-3T3) and a number of other cell lines,including human cells. In a particularly preferred embodiment, saidanimal cell is a CHO cell.

In a particularly preferred embodiment, said animal cell is a rabbitcell. Preferred insect cells include but are not limited to cells fromthe SF9 cell lines.

The antibody of the invention may be prepared by a method, wherein saidantibody is obtained from a host as described herein above. Thus, afurther embodiment of the present invention is a method for thepreparation of an antibody comprising culturing the host cell of theinvention under conditions that allow synthesis of said antibody andrecovering said antibody from said culture.

The transformed hosts can be grown in fermenters and cultured accordingto techniques known to those skilled in the art to achieve optimal cellgrowth. In addition, efficient expression and processing of newlysynthetized protein may depend on the presence of further amino aciddomains like signal peptides. In a further embodiment, antibodies of theinvention may comprise an N-terminal signal sequence, which allowssecretion from a host cell after recombinant expression. Although signalpeptides are heterogeneous, and many prokaryotic and eukaryotic signalpeptides are functionally interchangeable, the skilled person is awareof means to choose a suitable signal peptide according to the usedexpression system. Therefore, as a non-limiting example reference ismade to the signal peptide of SEQ ID NO: 52. Once expressed, the wholeantibodies, their dimers, individual light and heavy chains, or otherimmunoglobulin forms of the present invention can be purified accordingto standard procedures of the art, including ammonium sulphateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like; see Scopes, “Protein Purification”,Springer-Verlag, N. Y. (1982). The antibody or its correspondingimmunoglobulin chain(s) of the invention can then be isolated from thegrowth medium, cellular lysates or cellular membrane fractions. Theisolation and purification of the e.g. microbially expressed antibodiesor immunoglobulin chains of the invention may be by any conventionalmeans, such as for example preparative chromatographic separations andimmunological separations such as those involving the use of monoclonalor polyclonal antibodies directed e.g. against the constant region ofthe antibody of the invention.

It will be apparent to those skilled in the art that the antibodies ofthe invention can be further coupled to other moieties, e.g. drugtargeting and imaging applications, i.e. effector or labelling groups asdefined herein. Such coupling may be conducted chemically afterexpression of the antibody or antigen to side of attachment or thecoupling product may be engineered into the antibody or antigen of theinvention at the DNA level. The DNAs are then expressed in a suitablehost system, and the expressed proteins are collected and renatured ifnecessary.

According to one embodiment, a recombinant cell as described above iscultured under conditions which allow expression of the antibodyencoding nucleic acid molecules. The antibody may be collected from thecultured cell or the culture supernatant. Preferably, the antibody isprepared from a mammalian, particularly from a human cell. In anotherpreferred embodiment the antibody is prepared from CHO cells.

Still a further aspect of the present invention relates to apharmaceutical composition comprising the antibody as described above,optionally together with a pharmaceutically acceptable carrier.According to the invention, the pharmaceutical composition is adaptedfor a therapeutic use.

The term “carrier” includes agents, e.g. diluents, stabilizers,adjuvants or other types of excipients that are non-toxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Examples of pharmaceutically acceptable carriers are well-known in theart and include phosphate-buffered saline solutions, water, emulsionssuch as oil/water emulsions, various types of wetting agents, sterilesolutions, etc. Preferred examples of physiologically acceptablecarriers include buffers such as phosphate, citrate and other organicacids (however, with regard to the formulation of the present invention,a phosphate buffer is preferred); anti-oxidants including ascorbic acid,low molecular weight (less than about 10 residues) polypeptides;proteins such as serum albumin, gelatine or immunoglobulins; hydrophilicpolymers such as polyvinyl pyrrolidone; amino acids such as glycine,glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose ordextrins, chelating agents such as EDTA, sugar, alcohols such asmannitol or sorbitol; salt-forming counter ions such as sodium; and/ornon-ionic surfactants such as TWEEN, polyethylene or polyethyleneglycol.

The pharmaceutical composition may be formulated by well-knownconventional methods, i.e. by mixing the active agent with carriers andoptionally other agents that are usually incorporated into theformulation.

Another aspect of the present invention relates to a pharmaceuticalcomposition as described above, which contains at least one furtheractive agent. Which further active agent is used depends on theindication to be treated. For example, cytotoxic agents such asdoxorubicin, cisplatin or carboplatin, cytokines or otheranti-neoplastic agents may be used in the treatment of cancer. Inparticular for cancer treatment a combination with knownimmunotherapeutic agents in preferred. This combination includes but isnot limited to agents like: anti PD-1 antibodies, anti-PD-L1 antibodiesand anti-CTLA-4 antibodies.

According to a preferred embodiment the monoclonal antibody or thepharmaceutical composition according to the invention can be used toinhibit the CD95 signalling pathway. In particular, the antibody or thecomposition can be used in the prophylaxis and/or treatment of disordersselected from autoimmune disorders, AIDS, heart disorders, e.g.myocardial infarction, graft-versus-host disorders, transplantrejection, brain damage, e.g. stroke, spinal cord injuries, sepsis,hepatitis, NASH, disorders associated with inflammation, ischemicreperfusion injury and renal disorders. Of course, the compositiondescribed herein may be used for the treatment of cancers, preferablysolid cancers as well as lymphomas. Solid cancers comprise sarcomas andcarcinomas. For example, the cancer to be treated may be colon, lung,breast, pancreas, renal, colorectal, liver or brain cancers, e.g.,glioblastomas and/or metastasis thereof. Alternatively, the cancer to betreated may be a cancer of lymphoid or myeloid origin.

A further aspect of the present invention is a method of treatment ofcancer, said method comprising administering a monoclonal anti-CD95Lantibody of the present invention to a patient. For therapeutic use theCD95L antibody can be administered systemically, for example by infusionor injection.

The therapeutic method of the invention preferably includes a precedingstep of determining the expression of CD95L in a cancer sample obtainedfrom a patient. This diagnostic classification of cancer by the extentof CD95L expression enables an adapted therapy for those patientssuffering from a cancer expressing CD95L. It is preferred that themonoclonal anti-CD95L antibody of the invention is administered only ifexpression of CD95L has been detected in the cancer sample. Thisstrategy is advantageous, because the anti-CD95L antibody isadministered to those patients only in which a therapeutic success canbe expected. This is not disadvantageous in patients suffering from acancer not expressing CD95L, because these patients will probably notbenefit from a treatment with a CD95L inhibitor.

Expression of CD95L can be determined by any know method. For example,CD95L or CD95L mRNA can be determined. A preferred example of a suitablemethod is a histological, histochemical, immunohistochemical and/or flowcytometry based method. In particular, the expression of CD95L in thecancer sample can be determined by contacting the sample with an agentspecifically binding to CD95L. For example, CD95L inhibitorsspecifically binding to CD95L can be used for determination of CD95L.

Exemplary CD95L inhibitors include antibodies, soluble CD95 molecules,fusion proteins, etc. Suitable antibodies can be prepared by knownmethods. An example of a suitable antibody is a monoclonal antibody ofthe invention suitable for detection of CD95L in flow cytometry basedanalysis. Also preferred are anti-CD95L-specific antibodies or aCD95L-recognizing fragment thereof binding to an intracellular epitopeof CD95L. According to an especially preferred embodiment theanti-CD95L-specific antibody or CD95L-recognising fragment thereof bindsto tyrosine (Y) in N-terminal position 13 of human CD95L. According toespecially preferred embodiments a diagnostic anti-CD95L antibody or aCD95L-recognizing fragment thereof recognizes an epitope that includesthe N-terminal amino acids 13-19 of human CD95L. An example of asuitable antibody is described in WO 2014/177576.

According to an especially preferred embodiment of the invention, theantibodies described herein can be used for the determination of CD95Lexpression in a first step and if CD95L expression is detected, theinventive antibodies can be used for therapeutic purposes in a secondstep.

In the present invention, expression of CD95L is determined by any knownsuitable method, but using the antibody of the present invention. Forexample, the determination may comprise a histological, histochemicalimmunohistochemical (IHC) or/and flow cytometry based method using theabove-described anti-CD95L antibody. Immunohistochemical methods areparticularly preferred.

The sample employed in the classification of cancer as described hereincan be an archived tumour tissue, for example a biopsy or surgerymaterial embedded in paraffin, which has been obtained in an earlierstage of the disease.

The cancer disease can be classified by the level of CD95L expressioninto a CD95L positive cancer disease or a CD95L negative cancer disease.

In particular the CD95L positive cancer disease is characterised by acell expressing CD95L on the cell surface. However, the methodsdescribed herein may also be based on the detection of intracellularepitopes of CD95L.

A cancer can be regarded as CD95L positive, if at least 1%, at least 2%,at least 5%, at least 10%, at least 20%, or at least 50% of the cells ina cancer sample express CD95L. The number of CD95L positive cells can bedetermined by counting the cells in a microscopic section.

CD95L expression is considered to be absent (CD95L negative) ifessentially no cells expressing CD95L can be detected in the tissuesample, or if the sample is a sample which does not fulfil the criteriadefined herein for a CD95L positive sample (non-positive sample). In aCD95L negative sample, the number of tumour cells expressing CD95L canbe below the threshold defined herein for CD95L positive samples, forexample below 1%, below 2%, below 3%, below 4%, below 5%, or below 10%of tumour cells.

A cancer can also be regarded as CD95L positive, if CD95L can bedetected on at least 1%, at least 2%, at least 5%, at least 10%, atleast 20%, or at least 50% of the area of tumour tissue in a tissuesection. This value is termed herein as “% CD95L positive area of tumourtissue”. Non-tumour tissue is excluded in this analysis. A tissuesection can be prepared by known methods. Suitable methods for detectionof CD95L are described in WO 2014/177576. An exemplary method and anexample for the determination of the area of CD95L positive tumourtissue is given in WO 2014/177576. CD95L expression can be considered tobe absent (CD95L negative) if essentially no CD95L can be detected inthe tissue sample, or if the value of % CD95L positive area of tumourtissue is below the threshold defined for a CD95 positive sample, forexample below 1%, below 2%, below 3%, below 4%, below 5%, or below 10%of tumour area.

CD95L expression (e.g. in terms of cell number or surface in a tissuesection) can be determined by known methods, for example by methodsbased upon automatized analysis of tissue sections.

By the method of the present invention, any type of cancer, inparticular solid tumour tissue, can be diagnosed for expression ofCD95L. The cancer to be diagnosed or/and treated may also be a cancer oflymphoid or myeloid origin.

Diagnosis based upon the expression of CD95L is of particular importancefor diagnosis and treatment of those cancer types which include CD95Lexpression sub-types, and thus require a specific therapy adapted to thediagnosed CD95L expression sub-type. An example of CD95L expressionsub-types of glioblastoma identified in the present invention is CD95Lpositive glioblastoma and CD95L negative glioblastoma, as describedherein. The solution provided herein includes a specific therapy basedupon the diagnosis of the CD95L expression sub-types identified in thepresent invention.

Any type of cancer, in particular solid tumour tissue, can be determinedto be CD95L expression positive or CD95L expression negative. The cancercan be characterised by invasive growth. The cancer disease to bediagnosed according to the present invention as CD95L positive cancer orCD95L negative cancer can be selected from the group consisting of braincancer, colon cancer, colorectal cancer, pancreatic cancer, breastcancer, lung cancer, renal cancer, liver cancer or/and metastaticdisease thereof. In particular, the cancer disease is glioma, moreparticular glioblastoma.

For example, according to the present invention, the diagnosis braintumour by hitherto known diagnostic methods can be specified to be aCD95L positive brain tumour or a CD95L negative brain tumour, based uponthe outcome of determination of CD95L expression in a tumour sample, asdescribed herein. Such known diagnostic methods include knownhistological or histopathological methods such as known methods oftissue staining and known immunohistochemical methods.

Yet another aspect of the present invention is a monoclonal antibody ofthe invention for use in classifying a cancer disease according thelevel of CD95L expression. In this aspect, the cancer disease can beclassified by the level of CD95L expression into a CD95L positive cancerdisease or a CD95L negative cancer disease. The level of CD95Lexpression is preferably determined in an immunohistochemcial methodusing the antibody of the invention.

In this aspect, the cancer can be any cancer, as described herein. Inparticular, the cancer disease is selected from the group consisting ofbrain cancer, colon cancer, colorectal cancer, pancreatic cancer, breastcancer, lung cancer, renal cancer, liver cancer or/and metastaticdisease thereof. More particular, the cancer disease is glioma, mostparticular glioblastoma.

Another aspect of the present invention is an anti-CD95L antibody of theinvention for use in providing a prognosis about the overall survivaltime or/and the relapse-free survival time in a cancer patient, byclassifying the cancer disease of the patient by the level of CD95Lexpression. The level of CD95L expression is preferably determined in animmunohistochemcial method using the antibody of the invention.

In this aspect, the cancer can be any cancer, as described herein. Inparticular, the cancer disease is selected from the group consisting ofbrain cancer, colon cancer, colorectal cancer, pancreatic cancer, breastcancer, lung cancer, renal cancer, liver cancer or/and metastaticdisease thereof. More particular, the cancer disease is glioma, mostparticular glioblastoma.

In the present invention, the overall survival time (OS) denotes thechances of staying alive for a group of individuals suffering from acancer. It denotes the percentage of individuals in the group who arelikely to be alive after a particular duration of time.

Yet another aspect of the present invention is a method of providing aprognosis about the overall survival time or/and the relapse-freesurvival time in a cancer patient, said method comprising

-   (a) determining CD95L expression in a cancer sample using an    antibody of the invention, and-   (b) providing a prognosis about the survival time or/and the    relapse-free survival time of the patient by the level of CD95L    expression, wherein the CD95L expression is negatively correlated    with the survival time of the patient.

The invention is further illustrated by the following Figures andExamples.

FIGURE LEGENDS

FIG. 1: Peptide array with anti-CD95L antibody clone 119-4 (A) andintensity plot derived from said assay (B)

FIGS. 2A and 2B: Peptide array with anti-CD95L antibody clone 145-12 (A)and intensity plot derived from said assay (B)

FIGS. 3A and 3B: Peptide array with anti-CD96L antibody clone 103-7 (A)and intensity plot derived from said assay (B)

FIG. 4: Flow cytometric based detection of CD95L using the antibodies119-4, 145-12 and 103-7. Dashed histogram: rabbit isotype control;filled histogram: sub-clone supernatant

FIG. 5: Competition ELISA with specific competitor APG296

FIG. 6: Competition ELISA with non-specific competitor APG707

FIG. 7: Neutralization of APG101 binding to CD95L by subclones 119-4,103-7 and 145-12

FIG. 8: ELISA assessing the binding of subclones to CD95L from threedifferent species

FIG. 9: Biological activity of CD95L-blockers: antagonism of humanCD95L-induced apoptosis on Jurkat A3 cells

FIG. 10: Biological activity of CD95L-blockers: antagonism ofimmobilized human CD95L-induced apoptosis on Jurkat A3 cells

FIG. 11: Biological activity of CD95L-blockers: antagonism ofimmobilized monkey CD95L-induced apoptosis on Jurkat A3 cells

FIG. 12: Biological activity of CD95L-blockers: antagonism ofimmobilized mouse CD95L-induced apoptosis on Jurkat A3 cells

FIG. 13: Epitope mapping 119-4 peptide ELISA

FIG. 14: Epitope mapping 145-12 peptide ELISA

FIG. 15: Epitope mapping 103-7 peptide ELISA

FIG. 16: Determination of the K_(D) for antibodies 119-4 and 145-12

FIG. 17: Alignments of the amino acid sequences of the three humanizedvariable heavy (V_(H)) domains comprising either the original or themodified CDR-H's of rabbit monoclonal antibody 145-12 with the humanV_(H) consensus frameworks (hum III, heavy subgroup III) used.Complementarity Determining Regions (CDRs) are in brackets. The CDR'S ofthe recipient hum III are printed in italic and important heavy chainframework residues are marked (H28, H31a, H50, H71). All modificationsdescribed in the humanization procedure are printed in small letters andunderlined. Shown is Hum III of SEQ ID NO:53; huVH145_A of SEQ ID NO:30;huVH145_B of SEQ ID NO:31 and huVH145_C of SEQ ID NO:32

FIG. 18: Alignment of the amino acid sequences of the three humanizedvariable heavy (V_(H)) domains comprising either the original or themodified CDR-H's of rabbit monoclonal antibody 119-4 with the humanV_(H) consensus frameworks (hum III, heavy subgroup III) used.Complementarity Determining Regions (CDRs) are in brackets. The CDR'S ofthe recipient hum III are printed in italic and important heavy chainframework residues are marked (H28, H31a, H50, H71). All modificationsdescribed in the humanization procedure are printed in small letters andunderlined. Shown is Hum III of SEQ ID NO:53; huVH119_A of SEQ ID NO:41;huVH119_B of SEQ ID NO:42 and huVH119_C of SEQ ID NO:43

FIG. 19: Alignment of the amino acid sequences of humanized variablelight chain (V_(L)) domains of rabbit monoclonal antibodies 119-4 and154-12 and human V_(L) consensus framework. Complementarity DeterminingRegions (CDRs) are in brackets. The CDR'S of the recipient sequence areprinted in italic. Shown is hu_k1 of SEQ ID NO:54; hu119_4 of SEQ IDNO:44 and hu145_12 of SEQ ID NO:33

EXAMPLE 1: IMMUNIZATION/SCREENING STRATEGY FOR ANTI-CD95L

For the generation of CD95L-antibodies rabbits were immunised withrecombinant CD95L (APG296; SEQ ID NO: 26). Animals showing a high serumtiter against CD95L (APG296) by ELISA were selected for the generationof rabbit monoclonal antibodies. For this procedure lymphocytes wereisolated from rabbit spleen and fused with rabbit myeloma cells. Growinghybridoma cells were screened for the presence of antibodies in the cellculture supernatant and subsequently tested for their specificity torecognize CD95L. In summary, 163 supernatants of growing hybridoma weretested for detection of CD95L in an ELISA based assay as a primaryscreen. About 70 clones showed interaction with CD95L and were furthercharacterized in detail by ELISA, IHC, Western-Blot and FACS-analysis.Three clones (103, 119 and 145) were selected and sub-cloned via limiteddilution to ensure monoclonality. The (sub)clones 103-7, 119-4, 145-12were finally selected for further characterisation.

EXAMPLE 2: PEPTIDE ARRAY

Pre-staining of the peptide array was done with the secondary goatanti-rabbit IgG (H+L) DyLight680 antibody at a dilution of 1:5000 toinvestigate background interactions that could interfere with the mainassays. Subsequent incubation of the peptide microarrays with rabbitmonoclonal antibody clones 103-7, 119-4 and 145-12 at dilution of 1:1000and 1:100 (103-7 and 145-12) in incubation buffer was followed bystaining with the secondary goat anti-rabbit IgG (H+L) DyLight680antibody and read-out of the fluorescence intensities.

Quantification of spot intensities and peptide annotation were done withPepSlide® Analyzer and listed in an Excel file. A software algorithmbreaks down fluorescence intensities of each spot into raw, foregroundand background signal and calculates the standard deviation offoreground median intensities. Based on averaged foreground medianintensities, an intensity map was generated and binders in the peptidemap highlighted

The averaged spot intensities of the assays were plotted with rabbitmonoclonal antibodies 103-7, 119-4 and 145-12 against the human CD95Lsequence from the N- to the C-terminus to visualize overall spotintensities and signal to noise ratios (see FIGS. 1, 2 and 3). Theintensity plots were finally correlated with peptide and intensity mapsas well as with visual inspection of the microarray scans to identifythe peptides and consensus motif that interacted with the monoclonalantibody sample.

EXAMPLE 3: EPITOPE MAPPING OF RABBIT MONOCLONAL ANTIBODIES

Incubation of one of the peptide microarrays with rabbit monoclonalantibody 119-4 at a dilution of 1:1000 (left) was followed by stainingwith the secondary goat anti-rabbit IgG (H+L) DyLight680 antibody. Weobserved a strong and well-defined threefold spot pattern formed by rowsof neighboured peptides. This was in accordance with the microarraylayout shown in the peptide map with the 10 aa peptides on top, the 12aa peptides in the middle and the 15 aa peptides on bottom of thepeptide microarray.

Data quantification was followed by generation of peptide and intensitymaps as well as of an intensity plot. In accordance with the microarrayscan, we observed a strong and well-defined threefold epitope-like spotpattern after incubation with rabbit monoclonal antibody 119-4 at adilution of 1:1000 in incubation buffer. The rows of neighboured spotsat all peptide lengths were correlated with the consensus motif thatformed the epitope of rabbit monoclonal antibody 119-4 (FIG. 1). Similararrays were done for antibodies 145-12 and 103-7 (FIGS. 2 and 3).

For antibodies 119-4 and 145-12 a consensus epitope encoding the aminoacids RNSKYPQD could be assigned.

No epitope could be assigned for clone 103-7. The corresponding antibodyhas a non-linear structural epitope.

EXAMPLE 4: FACS ANALYSIS OF CD95L EXPRESSION

Flow cytometric analysis of CD95L expression was performed on KFL9 cell.Prior to the incubation with primary antibodies cells were blocked withFACS buffer (PBS, 5% FCS, 1/100 Gammunex). Subsequently, the primaryantibodies 103-7, 119-4 and 145-12 (or a respective isotype controlantibody) were added and incubated for 30 min. After three washing stepswith PBS a secondary goat anti-rabbit biotin antibody was added.Specifically, bound antibodies were detected by addition ofPE-conjugated Streptavidin. The entire protocol was performed on ice orat 4° C. Flow cytometric analysis was performed by a Guava EasyCyteMini. The histograms of FIG. 4 show the fluorescence intensity of theclones 103-7, 119-4 and 145-12 in comparison to the rabbit isotypecontrol antibody (dashed line). All clones are equally capable ofspecific detection of CD95L on the cell surface of KFL9 cells.

EXAMPLE 5: COMPETITION ELISA WITH SPECIFIC COMPETITOR APG296

For the competition ELISA, 96-well microtiter plates were coated with 10μg/ml APG296 (CD95L-RB69; SEQ ID NO: 26). After blocking withStartingBlock, wells were incubated with antibodies from subclones119-4, 103-7 and 145-12 at a final dilution of 1:200 in the absence orpresence of the specific competitor APG296 (0, 0.1, 1, 10 or 100 μg/ml).Binding of the rabbit monoclonal antibodies was detected by incubationwith goat anti rabbit IgG-Peroxidase (Sigma; dilution 1:5000) andsubsequent detection of the converted Peroxidase-substrate TMB one at awavelength of 450 nm in an ELISA reader (FIG. 5).

For antibodies from clones 119-4, 103-7 and 145-12 a dose-dependentcompetition of the ELISA signal was observed in the presence of thespecific competitor. At the highest concentration tested (100 μg/ml) theELISA signal was reduced to background level.

EXAMPLE 6: COMPETITION ELISA WITH NON-SPECIFIC COMPETITOR APG707

For the competition ELISA, 96-well microtiter plates were coated with 10μg/ml APG296 (CD95L-RB69). After blocking with StartingBlock, wells wereincubated with antibodies from subclones 119-4, 103-7 and 145-12 at afinal dilution of 1:200 in the absence or presence of the non-specificcompetitor APG707 (LIGHT-RB69; SEQ ID NO: 27; 0, 10 or 100 μg/ml).Binding of the rabbit monoclonal antibodies was detected by incubationwith goat anti rabbit IgG-Peroxidase (Sigma; dilution 1:5000) andsubsequent detection of the converted Peroxidase-substrate TMBone at awavelength of 450 nm in an ELISA reader (FIG. 6).

No competition was seen for antibodies from subclones 119-4, 103-7 and145-12 in the presence of an unspecific competitor. Even highconcentrations of APG707 showed no significant competition of the ELISAsignal.

EXAMPLE 7: NEUTRALIZATION OF CD95 (RECEPTOR) BINDING TO CD95L BYANTIBODIES FROM SUBCLONES 119-4. 103-7 AND 145-12

APG101 is a fusion protein comprising the Fc-part of human IgG1 and theextracelluar “Ligand Binding Domain” of CD95. APG101 shows strongbinding to CD95L and is particularly suited to analyse the ability ofCD95L-antibodies to interfere with CD95L/CD95 interaction:

The neutralization of the binding of APG101 to CD95L by subclones 119-4,103-7 and 145-12 was assessed by ELISA. 96-well microtiter plates werecoated with 5 μg/ml StrepMablmmo (IBA). After blocking withStartingBlock, wells were incubated with 1 μg/ml CD95L-T4 (APG293)containing a StrepTag which is captured by StrepMablmmo. Wells were thenincubated with subclones 119-4, 103-7 and 145-12 at dilutions of 1:10,1:50 and 1:250. In a next incubation step, APG101 at a concentration of1 μg/ml was added. Binding of APG101 to CD95L was detected by incubationwith goat anti human IgG-Peroxidase (Sigma; dilution 1:5000) andsubsequent detection of the converted Peroxidase-substrate TMBone at awavelength of 450 nm in an ELISA reader. Data are expressed as relativeELISA signal with a 100% value indicating no neutralisation of thebinding of APG101 to CD95L and a 0% value indicating a completeneutralization of the binding of APG101 to CD95L (FIG. 7).

Antibodies from subclones 103-7 and 145-12 showed neutralisation ofAPG101 binding in a dose dependent manner. In comparison antibodies fromsubclone 119-4 showed a more efficient neutralisation of APG101 binding.

EXAMPLE 8: ELISA ASSESSING THE BINDING OF ANTIBODY SUBCLONES TO CD95LFROM THREE DIFFERENT SPECIES

For the ELISA assessing the species specificity of three differentsubclones, 96-well microtiter plates were coated with 0.5 μg/ml humanCD95L-T4 (black) or 0.5 μg/ml macaca fascicularis CD95L-T4 (dark grey)or 0.5 μg/ml mus musculus CD95L-T4 (light grey). After blocking withStartingBlock, wells were incubated with subclones 119-4, 103-7 and145-12 at a final dilution of 1:200. Binding of the rabbit monoclonalantibodies was detected by incubation with goat anti rabbitIgG-Peroxidase (Sigma; dilution 1:5000) and subsequent detection of theconverted Peroxidase-substrate TMBone at a wavelength of 450 nm in anELISA reader (FIG. 8).

Clone 119-4 shows strong binding to CD95L from all tested species.Antibodies from clone 103-7 and 119-4 showed strong binding to human andmonkey CD95L and only weak binding to CD95L derived from mouse.

EXAMPLE 9: BIOLOGICAL ACTIVITY OF CD95L-BLOCKERS: ANTAGONISM OF HUMANCD95L-INDUCED APOPTOSIS ON JURKAT A3 CELLS

For the cellular assay assessing the biological activity of threedifferent subclones (103-7, 119-4, 145-12) in comparison to APG101,96-well microtiter plates were pipetted with 100000 Jurkat A3 cells perwell. Then, the wells were supplemented with a constant concentration offinally 250 ng/ml APG293 (human CD95L-T4; SEQ ID NO: 25) and a titrationof CD95L-antagonist as indicated on the x-axis. After 3 hours incubationat 37° C., cells were lysed with lysis buffer (250 mM HEPES, 50 mMMgCl2, 10 mM EGTA, 5% Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) andplates were put on ice for 30 minutes to 2 hours. Cleavage of thecaspase substrate Ac-DEVD-AFC was used to determine the extent ofapoptosis: 20 μl cell lysate was transferred to a black 96-wellmicrotiter plate; after the addition of 80 μl buffer containing 50 mMHEPES, 1% Sucrose, 0.1% CHAPS, 50 μM Ac-DEVD-AFC, and 25 mM DTT, pH 7.5,the plate was transferred to a Tecan microtiter plate reader and theincrease in fluorescence intensity was monitored (excitation 400 nm,emission 505 nm) (FIG. 9).

All four CD95L-antagonists show a dose-dependent inhibition of Caspaseinduction. The three antibodies (subclones 103-7, 119-4, 145-12) reveala higher antagonistic activity compared to APG101.

EXAMPLE 10: BIOLOGICAL ACTIVITY OF CD95L-BLOCKERS: ANTAGONISM OFAPOPTOSIS INDUCED ON JURKAT A3 CELLS BY IMMOBILIZED HUMAN CD95L

For the cellular assay assessing the biological activity of threedifferent subclones (103-7, 119-4, 145-12) in comparison to APG101,96-well StrepTactin microtiter plates (IBA) were incubated for 1 hourwith 250 ng/ml human CD95L-RB69 (APG296) which was captured by theimmobilised StrepTactin via its Strep-Tag. After washing the plate,CD95L-antagonists at different concentrations as indicated on the x-axiswere incubated for 1 hour. After washing, 100000 Jurkat A3 cells perwell were added. After 3 hours incubation at 37° C., cells were lysedwith lysis buffer (250 mM HEPES, 50 mM MgCl2, 10 mM EGTA, 5%Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) and plates were put onice for 30 minutes to 2 hours. Cleavage of the caspase substrateAc-DEVD-AFC was used to determine the extent of apoptosis: 20 μl celllysate was transferred to a black 96-well microtiter plate; after theaddition of 80 μl buffer containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS,50 μM Ac-DEVD-AFC, and 25 mM DTT, pH 7.5, the plate was transferred to aTecan microtiter plate reader and the increase in fluorescence intensitywas monitored (excitation 400 nm, emission 505 nm) (FIG. 10).

All tested antibodies showed efficient inhibition of apoptosis inducedby recombinant human CD95L (APG296). Compared to the knownCD95L-antagonist APG101 the antibodies showed a much higher efficacy.

EXAMPLE 11: BIOLOGICAL ACTIVITY OF CD95L-BLOCKERS: ANTAGONISM OFAPOPTOSIS INDUCED ON JURKAT A3 CELLS BY IMMOBILIZED MONKEY CD95L

For the cellular assay assessing the biological activity of threedifferent subclones (103-7, 119-4, 145-12) in comparison to APG101,96-well StrepTactin microtiter plates (IBA) were incubated for 1 hourwith 250 ng/ml monkey CD95L-RB69 (macaca fascicularis; APG1249) whichwas captured by the immobilised StrepTactin via its Strep-Tag. Afterwashing the plate, CD95L-antagonists at different concentrations asindicated on the x-axis were incubated for 1 hour. After washing, 100000Jurkat A3 cells per well were added. After 3 hours incubation at 37° C.,cells were lysed with lysis buffer (250 mM HEPES, 50 mM MgCl2, 10 mMEGTA, 5% Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) and plates wereput on ice for 30 minutes to 2 hours. Cleavage of the caspase substrateAc-DEVD-AFC was used to determine the extent of apoptosis: 20 μl celllysate was transferred to a black 96-well microtiter plate; after theaddition of 80 μl buffer containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS,50 μM Ac-DEVD-AFC, and 25 mM DTT, pH 7.5, the plate was transferred to aTecan microtiter plate reader and the increase in fluorescence intensitywas monitored (excitation 400 nm, emission 505 nm) (FIG. 11).

All tested antibodies showed efficient inhibition of apoptosis inducedby recombinant monkey CD95L (APG1249). Compared to the knownCD95L-antagonist APG101 the antibodies showed a much higher efficacy.

EXAMPLE 12: BIOLOGICAL ACTIVITY OF CD95L-BLOCKERS: ANTAGONISM OFAPOPTOSIS INDUCED ON JURKAT A3 CELLS BY IMMOBILIZED MOUSE CD95L

For the cellular assay assessing the biological activity of threedifferent subclones (103-7, 119-4, 145-12) in comparison to APG101,96-well StrepTactin microtiter plates (IBA) were incubated for 1 hourwith 250 ng/ml mouse CD95L-RB69 (mus musculus; APG1250) which wascaptured by the immobilised StrepTactin via its Strep-Tag. After washingthe plate, CD95L-antagonists at different concentrations as indicated onthe x-axis were incubated for 1 hour. After washing, 100000 Jurkat A3cells per well were added. After 3 hours incubation at 37° C., cellswere lysed with lysis buffer (250 mM HEPES, 50 mM MgCl2, 10 mM EGTA, 5%Triton-X-100, 100 mM DTT, 10 mM AEBSF, pH 7.5) and plates were put onice for 30 minutes to 2 hours. Cleavage of the caspase substrateAc-DEVD-AFC was used to determine the extent of apoptosis: 20 μl celllysate was transferred to a black 96-well microtiter plate; after theaddition of 80 μl buffer containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS,50 μM Ac-DEVD-AFC, and 25 mM DTT, pH 7.5, the plate was transferred to aTecan microtiter plate reader and the increase in fluorescence intensitywas monitored (excitation 400 nm, emission 505 nm).

All tested antibodies showed efficient inhibition of apoptosis inducedby recombinant mouse CD95L (APG1250). Compared to the knownCD95L-antagonist APG101 the antibodies showed a much higher efficacy.However, only the subclone 119-4 is able to reduce Caspase activityinduced by mouse CD95L to baseline levels (FIG. 12).

EXAMPLE 13: EPITOPE MAPPING 119-4 PEPTIDE ELISA

For the ELISA assessing the epitope of clone 119-4, 96-well microtiterplates were coated with 2 μg/ml human CD95L (APG296) or 2 μg/ml humanLIGHT (APG707) or peptides immobilized to BSA or ovalbumin that comprisea part of the extracellular amino acid sequence of human CD95L. Afterblocking with StartingBlock, wells were incubated with clone 119-4 at aconcentration of 2 μg/ml. Binding of the rabbit monoclonal antibody wasdetected by incubation with goat anti rabbit IgG-Peroxidase (Sigma;dilution 1:5000) and subsequent detection of the convertedPeroxidase-substrate TMBone at a wavelength of 450 nm in an ELISA reader(FIG. 13).

Antibody 119-4 shows binding to APG296 and to all tested peptides exceptof the peptide “C-YMRNSKY”. The respective binding pattern indicates aminimal epitope comprising the amino-acids “NSKYPQ”.

EXAMPLE 14: EPITOPE MAPPING 145-12 PEPTIDE ELISA

For the ELISA assessing the epitope of clone 145-12, 96-well microtiterplates were coated with 2 μg/ml human CD95L (APG296) or 2 μg/ml humanLIGHT (APG707) or peptides immobilized to BSA or ovalbumin that comprisea part of the extracellular amino acid sequence of human CD95L. Afterblocking with StartingBlock, wells were incubated with clone 145-12 at aconcentration of 2 μg/ml. Binding of the rabbit monoclonal antibody wasdetected by incubation with goat anti rabbit IgG-Peroxidase (Sigma;dilution 1:5000) and subsequent detection of the convertedPeroxidase-substrate TMBone at a wavelength of 450 nm in an ELISA reader(FIG. 14).

Antibody 145-12 shows specific binding to APG296. However, binding tothe tested peptides is weak or even absent (peptide “C-YMRNSKY”).Although the antibody shares the same epitope as clone 119-4 (as shownby peptide-array) it is conceivable that other possibly structuralcomponents of CD95L are required to define the full epitope of the145-12 antibody.

EXAMPLE 15: EPITOPE MAPPING 103-7 PEPTIDE ELISA

For the ELISA assessing the epitope of clone 103-7, 96-well microtiterplates were coated with 2 μg/ml human CD95L (APG296) or 2 μg/ml humanLIGHT (APG707) or peptides immobilized to BSA or ovalbumin that comprisea part of the extracellular amino acid sequence of human CD95L. Afterblocking with StartingBlock, wells were incubated with clone 103-7 at aconcentration of 2 μg/ml. Binding of the rabbit monoclonal antibody wasdetected by incubation with goat anti rabbit IgG-Peroxidase (Sigma;dilution 1:5000) and subsequent detection of the convertedPeroxidase-substrate TMBone at a wavelength of 450 nm in an ELISA reader(FIG. 15).

Antibody 103-7 shows specific binding to APG296. All peptide basedlinear epitopes are not detected by antibody 103-7, indicating anepitope that is defined by the three dimensional structure of CD95L.

EXAMPLE 16: DETERMINATION OF THE KO FOR ANTIBODIES 119-4 AND 145-12

The equilibrium binding constant (K) of antibodies 119-4 and 145-12 tothe epitope-comprising peptide “YMRNSKYPQD” was calculated based onkinetic binding data (k_(on) and k_(off)) determined with an automatedbiosensor system (Attana A100). The A100 allows to investigate molecularinteractions in real-time based on the Quartz Crystal Microbalance (QCM)technique. For this purpose, the respective epitope comprising peptidewas coupled to BSA and subsequently immobilized to the surface of acarboxyl-activated QCM-chip. Antibodies 119-4 and 145-12 were used assoluble analytes at different concentrations. Binding (k_(on)) anddissociation (k_(off)) was analyzed in real time, and the respectiveK_(D) was calculated (see table in FIG. 16).

Antibody 119-4 shows a higher affinity towards the epitope comprisingpeptide in comparison to antibody 145-12. K_(D) could not be analysedfor clone 103-7 with the chosen setup (data not shown).

Proteins Used for Immunization and Analysis:

The receptor-binding-domains of human, mouse and monkey CD95L(CD95L-RBD) were expressed as homotrimeric fusion proteins with aC-terminal positioned stabilization domain. Two versions of the humanCD95L-RBD were generated, identical regarding the CD95L-derivedsequence, but different in the molecular layout of the stabilizationdomain (APG293, SEQ ID NO: 25 and APG296, SEQ ID NO: 26). For theidentification and/or deselection of scaffold specific mAB's, astructural related protein from the TNF-superfamily comprising the sametrimerisation scaffold was used (APG707, SEQ ID NO: 27). For bindinganalysis, the monkey CD95L-RBD (APG1249, SEQ ID NO: 28) as well as mouseCD95L-RBD (APG1250, SEQ ID NO: 29) were expressed with the same fusionprotein technology. The general layout of aforementioned proteins andexamples for their production are described in U.S. Pat. No. 8,147,843B2and US008580273B2.

EXAMPLE 17: IN SILICO HUMANIZATION

In the following, residue numbering follows the Kabat enumeration. Forhumanization of the rabbit VH and VL antibody fragments derived from therabbit mAB 119-4 (SEQ ID NO:7 and SEQ ID NO:8) and mAB145-12 (SEQ IDNO:17 and SEQ ID NO:18) the following strategy was used: Instead ofsearching individual human VH/VL-germline sequences with high similarityto the individual donor VH/VL-rabbit sequences, a human recipient VH/VLdomain pair (VH subgroup III, SEQ ID 53 and VL kappa subgroup I SEQ ID54) was chosen, which was used frequently for the humanization procedureof murine VH/VL-domains as acceptor framework (Presta et al. 1997, Adamset al., 2006). For the humanization of the rabbit VL fragments of bothantibodies, a direct in silico grafting of the rabbit CDR-L's into thehuman VL-kappa subgroup I framework template (SEQ ID 54) without anychanges was performed (see FIG. 19). The resulting humanized VL domainof mAB145-12 has the SEQ ID 33 and the resulting humanized VL domain ofmAB119-4 has the SEQ ID 44.

For the humanization of the rabbit VH fragments of both antibodies, insilico grafting of the rabbit CDR-H's into the human framework templatewas performed and three humanized VH-sequence variants were created foreach donor rabbit-VH domain (see FIG. 17 and FIG. 18). In both humanizedvariant A sequences, positions H28, H71 and H73 were switched from therecipient (H28-T, H71-R, H73-N) to the donor sequence residues (H28-S,H71-K, H73-S). Variant A was the template for further modificationsresulting in variants B. In both humanized variant B sequences, inaddition to aforementioned mutation of the framework residues H28, H71and H73, the CDR-H2 positions H61, H62 and H63 of CDR-H2 were mutatedfrom the rabbit donor (H61-S, H62-W, H63-A) to the human acceptorresidues (H61-D, H62-S, H63-V). Variant B was the template for furthermodifications resulting in variants C. In both humanized variant Csequences, in addition to variant B mutations, the cysteine in therabbit CDR-H1 (position H35a) was mutated to serine and the cysteine inthe rabbit CDR-H2 (position H50) was mutated to alanine as cysteines arerare at those positions in human VH domains. The adjacent frameworkposition H49 in both humanized variant C sequences was mutated fromalanine to serine giving potentially a better structural support of themodified CDR-H2 in variants C. The resulting humanized VH domains ofmAB145-12 have the SEQ ID 30 (Variant A), SEQ ID 31 (Variant B) and SEQID 32 (Variant C). The resulting humanized VH domains of mAB119-4 havethe SEQ ID 41 (Variant A), SEQ ID 42 (Variant B) and SEQ ID 43 (VariantC).

In the case of the VH, Presta et al. defined the CDR-H1 by structuralaspects to comprise heavy chain residues H26-H35, whereas the sequencebased definition of the CDR-H1 comprises residues H31-H35 (Kabatenumeration). As the positions H26-H30 are involved in CDR-H1 loopconformation and position H28 is surface exposed, residue H28 is likelyto be mutated to the donor sequence. In addition, heavy chain positionsH69, H71 and H73 are known to be critical with respect to theconformation of the CDR-H loops of the VH subgroup III in general. Forthe chosen human VH/VL recipient domain pair it was discovered thatreplacement of the human to the donor residues was essential to enablefunctional engraftment of mouse CDR's in the aforementioned positions(Adams et al., 2006).

By visually inspecting the crystal structure of an Fab-fragment derivedfrom a monoclonal rabbit antibody (pdb entry 4ZT0, Chain H, SEQ ID 61),additional general features of the rabbit VH domain framework and therabbit CDR-H's were discovered. First of all, the CDR-H2 of the rabbitVH of SEQ ID 61 comprises C-terminal a tryptophane (residue H62-W) whichis anchoring a loop formed by residues H60-H65 positioned lateral at theVH-scaffold structure supporting the N-terminal CDR-H2 conformation atthe surface which is involved in antigen recognition. By its relativeposition close to the surface, this H62-tryptophane containing sequencemotif is likely to be potential immunogenic in a humanized antibodyintended for therapeutic purposes. A similar CDR-H2 loop-formingsequence is part of the mAb119-4 as well as of the mAb145-12. Therefore,we replaced the rabbit residues H61-H63 with the human residues H61-H63as described above to reduce the immunogenicity risk of the resultingantibody fragment in humans, as implemented in SEQ ID 31, SEQ ID 32, SEQID 42 and SEQ ID 43. An additional structural feature observed in theaforementioned structure is a disulfide-bridge formed by the rabbitresidues H35a and H50. Interestingly, this disulfide-bridge is buried inthe rabbit VH/VL domain interface and links two antiparallelbeta-barrels of the domain. As these beta-barrels support CDR-H1 andCDR-H2, a covalent linkage restricts potentially the structuralflexibility of the CDR-H1 and CDR-H2. This could lead to structuralfeatures enabling and/or enhancing binding of the recognized antigen. Asto proof this hypothesis for mAB119-4 and mAB145-12, in which the H35aand H50 cysteines are present, the human VH variants C (SEQ ID 32 andSEQ ID 43) were created where these cysteine residues were replaced.

EXAMPLE 18: FUNCTIONAL SCREENING OF THE HUMANIZED VH AND VL-DOMAINS OFm145-12 AND 119-4

For the compound based verification of the in silico humanizationprocedure, the scFv-minibody format was selected. HingelessscFv-minibodies containing the humanized VH/VL pairs presenting themAb145-12 and mAb119-4 specific CDR's were created according to Olafsenet al. 2004. The following modifications were implemented: The scFvswere generated in VH-VL orientation with a shorter 16 residue (GGGS)×4linker. The C-terminal serine of the human VH and the C-terminalarginine of the human VL are not present in the constructs. The CH3scaffold used comprises an N-terminal 5 residue linker element and aC-terminal Streptag-II for efficient affinity purification purposes atneutral pH. As a control the corresponding scFv-minibodies comprisingthe VH/VL domains of mAb 145-12 and mAb 119-4 were produced. In therabbit VL-domains, the singular cysteine forming the disulfide-bridge tothe rabbit kappa-constant domain was mutated to serine. For mammalianbased secretory pathway based production, synthetic cDNA-cassettes weregenerated encoding a suitable signal peptide in frame to thescFv-minibody of interest and cloned into expression vectors suitablefor stable expression in mammalian cells. Production of thescFv-minibodies was performed by the methods as described below. AllscFv-minibodies produced were finally purified by size exclusionchromatography ensuring multimer and aggregate depletion prior tofurther analytics, thereby excluding avidity effects in the subsequentactivity assays performed. The SEC-purified anti-CD95L specificscFv-minibodies were analysed for their capability to neutralize CD95Linduced apoptosis on Jurkat A3 cells. Functional reconstitution of theCD95L epitope recognition in the humanized scFv-Minibodies created withthe mAB145-12 or mAB119-4 CDR's is assumed to directly translate in EC50values comparable or lower than the EC50 values of the rabbit controlscFv-minibodies comprising the rabbit donor VH/VL-domains.

EXAMPLE 19: METHODS FOR CLONING, EXPRESSION AND PURIFICATION OFRECOMBINANT FULL LENGTH ANTIBODIES OR ANTIBODY FRAGMENTS

The aforementioned full length antibodies or antibody fragments areusually expressed recombinantly in two different eukaryotic host cells:

For initial analysis of aforementioned full length antibodies orantibody fragments, Hek293T cells grown in DMEM+GlutaMAX (GibCo)supplemented with 10% FBS, 100 units/ml Penicillin and 100 [mu]g/mlStreptomycin are transiently transfected with a plasmid containing anexpression cassette for recombinant polypeptide and an appropriateselection marker, e.g. a functional expression cassette comprising ablasticidine, puromycin or hygromycin resistence gene. In those cases,where a plurality of polypeptide chains is necessary to achieve thefinal product (e.g. full format antibodies), the expression cassettesare either combined on one plasmid or positioned on different plasmidsduring the transfection. Cell culture supernatant containing recombinantfusion polypeptides is harvested three days post transfection andclarified by centrifugation at 300×g followed by filtration through a0.22 μm sterile filter.

For larger scale expression of aforementioned full length antibodies orantibody fragments to be used in vivo, synthetic DNA cassettes encodingthe aforementioned proteins are inserted into eukaryotic expressionvectors comprising appropriate selection markers (e.g. a functionalexpression cassette comprising a blasticidin, puromycin or hygromycinresistance gene) and genetic elements suitable to enhance the number oftranscriptionally active insertion sites within the host cells genome,e.g the human β-globin matrix attachment region (MAR). The sequenceverified expression vectors are introduced by electroporation intosuspension adapted Chinese Hamster Ovary cells (CHO-S, Invitrogen).Appropriate selection pressure was applied three days post-transfectionto the transfected cells. Surviving cells carrying the vector derivedresistance gene(s) are recovered by subsequent cultivation underselection pressure. Upon stable growth of the selected cell pools inchemically defined medium (PowerCHO2-CD, Lonza) at 37° C. and 7% CO2atmosphere in an orbital shaker incubator (100 rpm, 50 mm shakingthrow), the individual supernatants are analyzed by ELISA-assaysdetecting the aforementioned proteins and the cell pools with thehighest specific productivity are expanded in shake flasks prior toprotein production (orbital shaker, 100 rpm, shaking throw 50 mm).

For lab-scale protein production, individual cell pools are cultured for7-12 days in chemically defined medium (PowerCHO2-CD, Lonza) at 37° C.and 7% CO2 atmosphere in a Wave bioreactor 20/50 EHT (GE-Healthcare).The basal medium is PowerCHO2-CD supplemented with 4 mM Glutamax. Waveculture started with a viable cell concentration of 0.3 to 0.4×10e6cells/ml and the following settings (for a five- or ten liter bag):shaking frequency 18 rpm, shaking ankle 7°, gas current 0.2-0.3 L/min,7% CO2, 36.5° C. During the Wave run, the cell culture are fed twicewith PowerFeed A (Lonza), usually on day 2 (20% feed) and day 5 (30%feed). After the second feed, shaking frequency is increased to 22 rpm,as well as the shaking ankle to 8°. The bioreactor is usually harvestedin between day 7 to day 12 when the cell viability dropped below 80%.First, the culture supernatant is clarified using a manual depthfiltration system (Millipore Millistak Pod, MC0HC 0.054 m2). ForStrep-tagged proteins, Avidin is added to a final concentration of 0.5mg/L. Finally, the culture supernatant containing the aforementionedfull length antibodies or antibody fragments is sterile filtered using abottle top filter (0.22 μm, PES, Corning) and stored at 2-8° C. untilfurther processing.

For affinity purification Streptactin Sepharose is packed to a column(gel bed 1 ml), equilibrated with 15 ml buffer W (100 mM Tris-HCl, 150mM NaCl, pH 8.0) or PBS pH 7.4 and the cell culture supernatant isapplied to the column with a flow rate of 4 ml/min. Subsequently, thecolumn is washed with 15 ml buffer W and bound polypeptide is elutedstepwise by addition of 7×1 ml buffer E (100 mM Tris HCl, 150 mM NaCl,2.5 mM Desthiobiotin, pH 8.0). Alternately, PBS pH 7.4 containing 2.5 mMDesthiobiotin can be used for this step.

Alternately to the Streptactin Sepharose based method, the affinitypurification is performed employing a column with immobilized Protein-Aas affinity ligand and a Akta chromatography system (GE-Healthcare). Asolid phase material with high affinity for the FC-domain of the fusionprotein is chosen: MABSelect Sure™ (GE Healthcare). Briefly, theclarified cell culture supernatant is loaded on a HiTrap MabSelectSurecolumn (CV=5 ml) equilibrated in wash-buffer-1 (20 mM Pi, 95 mM NaCl,pH7.2) not exceeding a load of 10 mg fusion protein per ml column-bed.The column is washed with ten column-volumes (10CV) of aforementionedequilibration buffer followed by four column-volumes (4CV) ofwash-buffer-2 (20 mM Pi, 95 mM NaCl, pH 8.0) to deplete host-cellprotein and host-cell DNA. The column is then eluted with elution buffer(20 mM Pi, 95 mM NaCl, pH 3.5) and the eluate is collected in up to tenfractions with each fraction having a volume equal to column-bed volume(5 ml). Each fraction is neutralized with an equal volume ofaforementioned wash-buffer-2. The linear velocity is set to 150 cm/h andkept constant during the aforementioned affinity chromatography method.

The protein amount of the eluate fractions is quantitated and peakfractions are concentrated by ultrafiltration and further purified bysize exclusion chromatography (SEC).

SEC is performed on Superdex 200 10/300 GL or HiLoad 26/60 columns usingan Akta chromatography system (GE-Healthcare). The columns areequilibrated with phosphate buffered saline and the concentrated,affinity-purified polypeptide is loaded onto the SEC column with thesample volume not exceeding 2% (v/v) of the column-volume. In the caseof Superdex 200 10/300 GL columns (GE Healthcare), a flow rate of 0.5 mlper minute is applied. In the case of HiLoad 26/60 Superdex200 columns,a flow rate of 2.5 ml per minute is applied. The elution profile of thepolypeptide is monitored by absorbance at 280 nm.

EXAMPLE 20: POTENCY OF CD95L BLOCKERS

Standard potency assay, according to Example 9, was used to analyse theantagonistic CD95L activity of humanized VH/VL-domains in thescFv-Minibody format. Functional reconstitution of the CD95L epitoperecognition in the humanized scFv-Minibodies created with the mAB145-12or mAB119-4 CDR's is assumed to directly translate in EC50 valuescomparable or lower than the EC50 values of the rabbit controlscFv-minibodies (SEC ID 37 representing mAb145-12 specificity and SEQ ID48 representing mAb119-4 specificity). The chosen humanization strategyallowed to preserve activity in the range of the initial rabbit antibodyas represented by the unmodified CDRs examplified in the scFv-minibodieswith SEQ ID 38 and 49. Surprisingly, the deimmunisation of the CDR-H2 ofmAB145-12 as examplified in SEQ ID 39 increased potency. Thedeimmunisation of the CDRH2 of mAb119-4 worked also, as the relativeactivity of scFv-minibody SEQ ID50 is comparable compared to thescFV-minibody with the SEQ ID 49. In contrast, modifications in of H35ain CDRH1 and H50 in CDRH2 significantly decreased potency asdemonstrated with the SEQ ID 40 and SEQ ID 51 based scFv-minibodies.

Compound Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 Mean STABWAPG101 774 789 707 714 — — 746 42 maB145-12 187 198 225 192 — — 201 16.9SEQ ID 37 124 117 — — 113 118 118 4.7 SEQ ID 38 118 114 — — 95.7 111 1109.7 SEQ ID 39 99.4 105 — — 86.5 83.4 93 10.2 SEQ ID 40 532 591 — — 701571 599 72.5 SEQ ID 48 — — 116 90.8 84.4 97.5 97 13.8 SEQ ID 49 — — 172144 117 127 140 24 SEQ ID 50 — — 169 136 128 140 143 18 SEQ ID 51 — —1549 2256 1479 1737 1755 351

Table shows biological in vitro activity of different CD95L neutralizingreagents. Activity is determined as the antagonizing activity of thecompounds with respect to the apoptosis induction of 250 ng/ml solubleCD95L-T4 on Jurkat A3 cells. Apoptosis induction is measured as cleavageof the substrate Ac-DEVD-AFC by Caspase 3/7. Values are expressed asEC50 in ng/ml.

EXAMPLE 21: GENERATION OF FULL LENGTH ANTIBODY FORMATS

Full length human antibody formats can be generated by fusing thehumanized VH and VL domains on appropriate scaffolds comprising theantibodies constant regions. An appropriate example sequence for thehuman constant kappa light chain is given in SEQ ID 58. Appropriateexample sequences for the IGG1 constant heavy chain regions are given inSEQ ID 59 and SEQ ID 60. As an example, fusing humanizd VL of mAb145-12(SEQ ID 33) to the kappa constant light chain results in SEQ ID 36representing a full length kappa light chain suitable to generate fullformat human antibodies with mAb145-12 specificity. Accordingly, byfusing humanized VL of mAb119-4 (SEQ ID NO 44) to the kappa constantlight chain SEQ ID 58 results in SEQ ID 47 representing a full lengthkappa light chain suitable to generate full format human antibodies withmAb119-4 specificity. Similarly, the necessary human heavy chains arecreated by fusing SEQ ID 42 with SEQ ID 59 or SEQ ID 60 resulting in afull length human heavy chains (SEQ ID 46 and SEQ ID 45) suitable forthe generation of full format human antibodies with mAb119-4specificity.

Accordingly, fusing SEQ ID 31 with SEQ ID 59 or SEQ ID 60 results in afull length human heavy chains (SEQ ID 35 and SEQ ID 34) suitable forthe generation of full format human antibodies with mAb145-12specificity. Expression technologies to produce full format recombinantantibodies in mammalian cell culture are well established in the art.

For those ordinary skilled in the art, it is obvious that that otherantibody scaffold technologies can be applied by employing the humanizedVH/VL domains to generate different formats with the desired antibodyspecificity.

1. A monoclonal anti-CD95L antibody characterized in that the antibodyspecifically binds to an epitope of human CD95L comprising the aminoacid sequence RNSKYP, preferably RNSKYPQ or RNSKYPQD, and inhibits CD95Linduced signalling.
 2. The monoclonal antibody according to claim 1,which specifically binds to CD95L derived from different species, inparticular to human, and at least one of monkey and mouse CD95L.
 3. Themonoclonal antibody according to claim 1, which specifically interactswith the Receptor Binding Domain of CD95L (on the cell surface andsoluble CD95L).
 4. The monoclonal antibody according to claim 1, whichis a full-length immunoglobulin or a functional immunoglobulin fragmentselected from the group consisting of Fab, Fab′, F(ab′)2, Fv, singlechain antibodies (scFv) and single domain antibodies.
 5. The monoclonalantibody according to claim 1, comprising a heavy chain amino acidsequence including CDRH1 as shown in SEQ ID NO: 1 or 11, CDRH2 as shownin SEQ ID NO: 2 or 12, and CDRH3 as shown in SEQ ID NO: 3 or 13, and alight chain amino acid sequence including CDRL1 as shown in SEQ ID NO: 4or 14, CDRL2 as shown in SEQ ID NO: 5 or 15, and CDRL3 as shown in SEQID NO: 6 or
 16. 6. The monoclonal antibody according to claim 5,comprising (i) a heavy chain amino acid sequence comprising CDRH1 asshown in SEQ ID NO: 1, CDRH2 as shown in SEQ ID NO: 2 and CDRH3 as shownin SEQ ID NO: 3 and a light chain amino acid sequence comprising CDRL1as shown in SEQ ID NO: 4, CDRL2 as shown in SEQ ID NO: 5 and CDRL3 asshown in SEQ ID NO: 6 or (ii) a heavy chain amino acid sequencecomprising CDRH1 as shown in SEQ ID NO: 11, CDRH2 as shown in SEQ ID NO:12 and CDRH3 as shown in SEQ ID NO: 13 and a light chain amino acidsequence comprising CDRL1 as shown in SEQ ID NO: 14, CDRL2 as shown inSEQ ID NO: 15 and CDRL3 as shown in SEQ ID NO:
 16. 7. The monoclonalantibody according to claim 1, comprising at least a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 7 or 17,and a light chain variable region having the amino acid sequence of SEQID NO: 8 or 18, or an amino acid sequence having a sequence identity ofat least 90% thereto.
 8. The monoclonal antibody according to claim 1,comprising a heavy chain amino acid sequence of SEQ ID NO: 9 or 19, anda light chain amino acid sequence of SEQ ID NO: 10 or 20, or an aminoacid sequence having a sequence identity of at least 90% thereto.
 9. Themonoclonal antibody according to claim 1, which is a humanized or humanantibody.
 10. The monoclonal antibody according to claim 9, comprisingat least a heavy chain variable region having the amino acid sequence ofSEQ ID NO: 30 or 31 or 32, and a light chain variable region having theamino acid sequence of SEQ ID NO: 33, or an amino acid sequence having asequence identity of at least 90% thereto.
 11. The monoclonal antibodyaccording to claim 9, comprising a heavy chain variable region havingthe amino acid sequence of SEQ ID NO: 30 or 31 or 32 fused to human IGG1heavy chain scaffold of SEQ ID NO: 59 or SEQ ID NO: 60, and a lightchain amino acid sequence of SEQ ID NO: 36, or an amino acid sequencehaving a sequence identity of at least 90% thereto.
 12. The monoclonalantibody according to claim 9, comprising a heavy chain amino acidsequence of SEQ ID NO: 34 or 35, and a light chain amino acid sequenceof SEQ ID NO: 36, or an amino acid sequence having a sequence identityof at least 90% thereto.
 13. The monoclonal antibody according to claim9, comprising at least a heavy chain variable region having the aminoacid sequence of SEQ ID NO: 41, 42 or 43, and a light chain variableregion having the amino acid sequence of SEQ ID NO: 44, or an amino acidsequence having a sequence identity of at least 90% thereto.
 14. Themonoclonal antibody according to claim 9, comprising a heavy chainvariable region having the amino acid sequence of SEQ ID NO: 41 or 42 or43 fused to human IGG1 heavy chain scaffold of SEQ ID NO: 59 or SEQ IDNO: 60, and a light chain amino acid sequence of SEQ ID NO: 47, or anamino acid sequence having a sequence identity of at least 90% thereto.15. The monoclonal antibody according to claim 9, comprising a heavychain amino acid sequence of SEQ ID NO: 45 or 46, and a light chainamino acid sequence of SEQ ID NO: 47, or an amino acid sequence having asequence identity of at least 90%.
 16. A monoclonal single chainantibody having the sequence selected of SEQ ID NO: 37, 38, 39, 40, 48,49, 50 or
 51. 17. The monoclonal antibody according to claim 1, whereina label or effector group is covalently attached to the antibody.
 18. Anisolated nucleic acid molecule comprising a nucleic acid sequenceselected from the group consisting of: (a) a nucleic acid sequenceencoding an antibody according to claim 1, (b) a nucleic acid sequencecomplementary to the sequences in (a), and (c) a nucleic acid sequencecapable of hybridizing to (a) or (b) under stringent conditions.
 19. Theisolated nucleic acid molecule according to claim 18, comprising thenucleic acid sequences as shown in SEQ ID NOs: 21 and 22 or as shown inSEQ ID NOs: 23 and
 24. 20. A vector comprising the nucleic acid sequenceas defined in claim 18.